In this paper, a highly conservedpiggyBac-like sequence, designated as McrPLE was cloned from a lepidopteran insect, Macdunnoughia crassisigna. It is 2 472 bp long in full length with a single open reading frame and e...In this paper, a highly conservedpiggyBac-like sequence, designated as McrPLE was cloned from a lepidopteran insect, Macdunnoughia crassisigna. It is 2 472 bp long in full length with a single open reading frame and encodes a 595 amino acid transposase. It shares identical terminal and sub-terminal repeats with T. ni IFP2 and is flanked by the typical TTAA target-site duplications. Alignment and phylogenetic analysis revealed that McrPLE had greater than 99.5% identity and appeared to be the closest one in phylogeny to IFP2 among the PLEs so far found in various species. Plasmid-based excision and transposition assay proved it was mobile in cell culture. Otherwise, McrPLE element and all other highly conserved IFP2 sequences reported previously were found to share three common nucleotide substitutions. This suggests that the original IFP2 may be a related variant of a predecessor element that became widespread. The existence of nearly identical piggyBac sequence in reproductively isolated species was thought also a strong indication of horizontal transmission, which raises important considerations for the stability and practical use ofpiggyBac transformation vectors.展开更多
Nine piggyBac-like elements （PLEs） were identified from the cotton aphid Aphis gossypii Glover. All the PLEs shared high sequence similarity with each other. However, eight of the nine PLEs were unlikely to encode f...Nine piggyBac-like elements （PLEs） were identified from the cotton aphid Aphis gossypii Glover. All the PLEs shared high sequence similarity with each other. However, eight of the nine PLEs were unlikely to encode functional transposase due to the existence of disruptive mutations within the coding regions. The other one PLE contained major characteristics of members in the piggyBac family, including TTAA target site duplications, inverted terminal repeats （ITRs）, and an open reading frame （ORF） coding for a transposase with a putative DDD domain. This one with an intact transposase ORF was named AgoPLE1.1. The predicted transposase shared 47% similarity with that of Trichoplusia ni piggyBac IFP2. Phylogenetic analyses showed that AgoPLE1.1 was most related to the Heliothis virescens PLE1.1 （HvPLE1.1） element, with 45% and 60% similarity at the nucleotide and amino acid levels, respectively. A functional assay demonstrated that AgoPLE1.1 encoded a functional transposase and was able to cause precise excision in cell cultures. On the other hand, few genomic insertion polymorphisms of AgoPLEI were observed in the genome of the cotton aphid. These observations suggested that AgoPLE1.1 was a PLE that invaded the cotton aphid genome in recent periods and retained its activity.展开更多
Piggy Bac is a transposable DNA element originally discovered in the cabbage looper moth(Trichoplusia ni).The T.ni piggyBac transposon can introduce exogenous fragments into a genome,constructing a transgenic organism...Piggy Bac is a transposable DNA element originally discovered in the cabbage looper moth(Trichoplusia ni).The T.ni piggyBac transposon can introduce exogenous fragments into a genome,constructing a transgenic organism.Nevertheless,the comprehensive analysis of endogenous piggyBac-like elements(PLEs)is important before using piggyBac,because they may influence the genetic stability of transgenic lines.Herein,we conducted a genome-wide analysis of PLEs in the brown planthopper(BPH)Nilaparvata lugens(St?l)(Hemiptera:Delphacidae),and identified a total of 28 PLE sequences.All N.lugens piggy Bac-like elements(NlPLEs)were present as multiple copies in the genome of BPH.Among the identified NlPLEs,NlPLE25 had the highest copy number and it was distributed on five chromosomes.The full length of NlPLE25 consisted of terminal inverted repeats and sub-terminal inverted repeats at both terminals,as well as a single open reading frame transposase encoding546 amino acids.Furthermore,NlPLE25 transposase caused precise excision and transposition in cultured insect cells and also restored the original TTAA target sequence after excision.A cross-recognition between the NlPLE25 transposon and the piggy Bac transposon was also revealed in this study.These findings provide useful information for the construction of transgenic insect lines.展开更多
To express human insulin-like growth factor-I (hIGF-I) in transformed Bombyx mori cultured cells and silk glands, the transgenic vector pigA3GFP-hIGF-ie-neo was constructed with a neomycin resistance gene driven by th...To express human insulin-like growth factor-I (hIGF-I) in transformed Bombyx mori cultured cells and silk glands, the transgenic vector pigA3GFP-hIGF-ie-neo was constructed with a neomycin resistance gene driven by the baculovirus ie-1 promoter, and with the hIGF-I gene under the control of the silkworm sericin promoter Ser-1. The stably transformed BmN cells expressing hIGF-I were selected by using the antibiotic G418 at a final concentration of 700-800 μg/mL after the BmN cells were transfected with the piggyBac vector and the helper plasmid. The specific band of hIGF-I was detected in the transformed cells by Western blot. The expression level of hIGF-I, determined by ELISA, was about 7800 pg in 5×105 cells. Analysis of the chromosomal insertion sites by inverse PCR showed that exogenous DNA could be inserted into the cell genome randomly or at TTAA target sequence specifically for piggyBac element transposition. The transgenic vector pigA3GFP-hIGF-ie-neo was transferred into the eggs using sperm-mediated gene transfer. Finally, two transgenic silkworms were obtained after screening for the neo and gfp genes and verified by PCR and dot hybridization. The expression level of hIGF-I determined by ELISA was about 2440 pg/g of silk gland of the transgenic silkworms of the G1 generation.展开更多
文摘In this paper, a highly conservedpiggyBac-like sequence, designated as McrPLE was cloned from a lepidopteran insect, Macdunnoughia crassisigna. It is 2 472 bp long in full length with a single open reading frame and encodes a 595 amino acid transposase. It shares identical terminal and sub-terminal repeats with T. ni IFP2 and is flanked by the typical TTAA target-site duplications. Alignment and phylogenetic analysis revealed that McrPLE had greater than 99.5% identity and appeared to be the closest one in phylogeny to IFP2 among the PLEs so far found in various species. Plasmid-based excision and transposition assay proved it was mobile in cell culture. Otherwise, McrPLE element and all other highly conserved IFP2 sequences reported previously were found to share three common nucleotide substitutions. This suggests that the original IFP2 may be a related variant of a predecessor element that became widespread. The existence of nearly identical piggyBac sequence in reproductively isolated species was thought also a strong indication of horizontal transmission, which raises important considerations for the stability and practical use ofpiggyBac transformation vectors.
基金Acknowledgments We thank Professor T. A. Miller from the University of California, Riverside, CA for his kindness in providing the helper pBacHsp and donor pB[KOα] plasmid. This work was supported by a national project for basic research （2006CB 102003）, China postdoctoral science foundation （20090451226）, and IAEA project （15686/RO/RBF）.
文摘Nine piggyBac-like elements （PLEs） were identified from the cotton aphid Aphis gossypii Glover. All the PLEs shared high sequence similarity with each other. However, eight of the nine PLEs were unlikely to encode functional transposase due to the existence of disruptive mutations within the coding regions. The other one PLE contained major characteristics of members in the piggyBac family, including TTAA target site duplications, inverted terminal repeats （ITRs）, and an open reading frame （ORF） coding for a transposase with a putative DDD domain. This one with an intact transposase ORF was named AgoPLE1.1. The predicted transposase shared 47% similarity with that of Trichoplusia ni piggyBac IFP2. Phylogenetic analyses showed that AgoPLE1.1 was most related to the Heliothis virescens PLE1.1 （HvPLE1.1） element, with 45% and 60% similarity at the nucleotide and amino acid levels, respectively. A functional assay demonstrated that AgoPLE1.1 encoded a functional transposase and was able to cause precise excision in cell cultures. On the other hand, few genomic insertion polymorphisms of AgoPLEI were observed in the genome of the cotton aphid. These observations suggested that AgoPLE1.1 was a PLE that invaded the cotton aphid genome in recent periods and retained its activity.
基金the Foundation of Guangzhou Science and Technology Key Project(No.201904020041),China。
文摘Piggy Bac is a transposable DNA element originally discovered in the cabbage looper moth(Trichoplusia ni).The T.ni piggyBac transposon can introduce exogenous fragments into a genome,constructing a transgenic organism.Nevertheless,the comprehensive analysis of endogenous piggyBac-like elements(PLEs)is important before using piggyBac,because they may influence the genetic stability of transgenic lines.Herein,we conducted a genome-wide analysis of PLEs in the brown planthopper(BPH)Nilaparvata lugens(St?l)(Hemiptera:Delphacidae),and identified a total of 28 PLE sequences.All N.lugens piggy Bac-like elements(NlPLEs)were present as multiple copies in the genome of BPH.Among the identified NlPLEs,NlPLE25 had the highest copy number and it was distributed on five chromosomes.The full length of NlPLE25 consisted of terminal inverted repeats and sub-terminal inverted repeats at both terminals,as well as a single open reading frame transposase encoding546 amino acids.Furthermore,NlPLE25 transposase caused precise excision and transposition in cultured insect cells and also restored the original TTAA target sequence after excision.A cross-recognition between the NlPLE25 transposon and the piggy Bac transposon was also revealed in this study.These findings provide useful information for the construction of transgenic insect lines.
基金Supported by the National Basic Research Program of China (Grant No. 2005CB121000)the National Natural Science Foundation of China (Grant No. 30671590)
文摘To express human insulin-like growth factor-I (hIGF-I) in transformed Bombyx mori cultured cells and silk glands, the transgenic vector pigA3GFP-hIGF-ie-neo was constructed with a neomycin resistance gene driven by the baculovirus ie-1 promoter, and with the hIGF-I gene under the control of the silkworm sericin promoter Ser-1. The stably transformed BmN cells expressing hIGF-I were selected by using the antibiotic G418 at a final concentration of 700-800 μg/mL after the BmN cells were transfected with the piggyBac vector and the helper plasmid. The specific band of hIGF-I was detected in the transformed cells by Western blot. The expression level of hIGF-I, determined by ELISA, was about 7800 pg in 5×105 cells. Analysis of the chromosomal insertion sites by inverse PCR showed that exogenous DNA could be inserted into the cell genome randomly or at TTAA target sequence specifically for piggyBac element transposition. The transgenic vector pigA3GFP-hIGF-ie-neo was transferred into the eggs using sperm-mediated gene transfer. Finally, two transgenic silkworms were obtained after screening for the neo and gfp genes and verified by PCR and dot hybridization. The expression level of hIGF-I determined by ELISA was about 2440 pg/g of silk gland of the transgenic silkworms of the G1 generation.
